ABSTRACT: Human induced pluripotent stem cells (hiPSCs) hold substantial promise for regenerative medicine and cell manufacturing, yet their widespread adoption remains limited by reprogramming strategies that are inefficient, costly, unsafe, and difficult to scale, driven by reliance on viral vectors, limited virus-free gene delivery technologies, and two-dimensional (2D) culture systems lacking microenvironmental control. Here, we present a virus-free, materials-guided reprogramming platform that integrates engineered polymeric nanoparticles with a biomimetic three-dimensional (3D) scaffold to jointly regulate gene delivery and cell-state transitions during fate conversion. A rationally designed fluorocarbon- and heparin-modified polymer nanoparticle enables efficient, low-toxicity episomal delivery of reprogramming factors, while a porous chitosan scaffold provides a permissive 3D microenvironment that accelerates reprogramming kinetics and suppresses somatic cell overgrowth. This integrated strategy concurrently improves reprogramming efficiency, kinetics, and workflow simplicity without the use of viral vectors or manual colony isolation. Scaffold-supported reprogramming yields an approximately eightfold increase in SSEA-4⁺/TRA-1-60⁺ cells by day 25 compared with non-viral 2D conditions, with pluripotent populations emerging as early as day 7, enabling hiPSC generation within weeks rather than the months typically required by conventional workflows. Transcriptomic analyses reveal that the 3D scaffold functions as an active regulatory element, suppressing inflammatory and extracellular matrix–associated programs that stabilize somatic identity while promoting chromatin-remodeling and stem-cell-associated pathways. Importantly, the platform supports a continuous, selection-free workflow, enabling direct differentiation of hiPSCs within the scaffold and further reducing processing time. By coupling delivery chemistry with microenvironmental control, this nanoparticle–scaffold platform provides a safe, efficient, and scalable route for hiPSC generation with direct relevance to regenerative medicine and patient-specific cell manufacturing.
